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|Application||This EZChIP kit contains all necessary reagents to perform 22 individual chromatin immunoprecipitation (ChIP) reactions using inexpensive protein G agarose beads. Control primers included.|
|Safety Information according to GHS|
|Storage and Shipping Information|
|Storage Conditions||Upon receipt, store components at the temperatures indicated on the labels. Kit components are stable for 1 year from date of shipment when stored as directed.|
|Material Size||22 assays|
|Material Package||Kit capacity: 22 chromatin immunoprecipitation assays|
|Reference overview||Application||Species||Pub Med ID|
|FOXQ1 controls the induced differentiation of melanocytic cells.|
Bagati, A; Bianchi-Smiraglia, A; Moparthy, S; Kolesnikova, K; Fink, EE; Kolesnikova, M; Roll, MV; Jowdy, P; Wolff, DW; Polechetti, A; Yun, DH; Lipchick, BC; Paul, LM; Wrazen, B; Moparthy, K; Mudambi, S; Morozevich, GE; Georgieva, SG; Wang, J; Shafirstein, G; Liu, S; Kandel, ES; Berman, AE; Box, NF; Paragh, G; Nikiforov, MA
Cell Death Differ 25 1040-1049 2018
Oncogenic transcription factor FOXQ1 has been implicated in promotion of multiple transformed phenotypes in carcinoma cells. Recently, we have characterized FOXQ1 as a melanoma tumor suppressor that acts via repression of N-cadherin gene, and invasion and metastasis. Here we report that FOXQ1 induces differentiation in normal and transformed melanocytic cells at least partially via direct transcriptional activation of MITF gene, melanocytic lineage-specific regulator of differentiation. Importantly, we demonstrate that pigmentation induced in cultured melanocytic cells and in mice by activation of cAMP/CREB1 pathway depends in large part on FOXQ1. Moreover, our data reveal that FOXQ1 acts as a critical mediator of BRAFV600E-dependent regulation of MITF levels, thus providing a novel link between two major signal transduction pathways controlling MITF and differentiation in melanocytic cells.
|DeSUMOylation switches Kaiso from activator to repressor upon hyperosmotic stress.|
Zhenilo, S; Deyev, I; Litvinova, E; Zhigalova, N; Kaplun, D; Sokolov, A; Mazur, A; Prokhortchouk, E
Cell Death Differ 0 2018
Kaiso is a member of the BTB/POZ zinc finger family, which is involved in cancer progression, cell cycle control, apoptosis, and WNT signaling. Depending on promoter context, it may function as either a transcriptional repressor or activator. Previous studies found that Kaiso might be SUMOylated due to heat shock, but the biological significance of Kaiso SUMOylation is unclear. Here, we find that K42 is the only amino acid within Kaiso that is modified with SUMO. Kaiso is monoSUMOylated at lysine 42 in cell lines of kidney origin under normal physiological conditions. SUMOylated Kaiso can activate transcription from exogenous methylated promoters, wherein the deSUMOylated form of the protein kept the ability to be a repressor. Rapid Kaiso deSUMOylation occurs in response to hyperosmotic stress and is reversible upon return to an isotonic environment. DeSUMOylation occurs within minutes in HEK293 cells treated with 100 mM NaCl and relaxes in 3 h even in a salt-containing medium. Genomic editing of Kaiso by conversion of K42 into R42 (K42R) in HEK293 cells that resulted in fully deSUMOylated endogenous protein led to misregulation of genes associated with ion transport, blood pressure, and the immune response. TRIM25 was significantly repressed in two K42R HEK293 clones. By a series of rescue experiments with K42R and KO HEK293 cells, we show that TRIM25 is a direct transcriptional target for Kaiso. In the absence of Kaiso, the level of TRIM25 is insensitive to hyperosmotic stress. Extending our observations to animal models, we show that in response to a high salt diet, Kaiso knockout mice are characterized by significantly higher blood pressure increases when compared to wild-type animals. Thus, we propose a novel biological role for Kaiso in the regulation of homeostasis.
|Alcohol dysregulates miR-148a in hepatocytes through FoxO1, facilitating pyroptosis via TXNIP overexpression.|
Heo, MJ; Kim, TH; You, JS; Blaya, D; Sancho-Bru, P; Kim, SG
Gut 0 2018
Alcoholic liver disease (ALD) is a leading cause of death among chronic liver diseases. However, its pathogenesis has not been completely established. MicroRNAs (miRNAs) are key contributors to liver diseases progression. This study investigated hepatocyte-abundant miRNAs dysregulated by ALD, its impact on hepatocyte injury and the underlying basis.Alcoholic hepatitis (AH) human and animal liver samples and hepatocytes were used to assess miR-148a levels. Pre-miR-148a was delivered specifically to hepatocytes in vivo using lentivirus. Immunoblottings, luciferase reporter assays, chromatin immunoprecipitation and immunofluorescence assays were carried out in cell models.The miRNA profile and PCR analyses enabled us to find substantial decrease of miR-148a in the liver of patients with AH. In mice subjected to Lieber-DeCarli alcohol diet or binge alcohol drinking, miR-148a levels were also markedly reduced. In cultured hepatocytes and mouse livers, alcohol exposure inhibited forkhead box protein O1 (FoxO1) expression, which correlated with miR-148a levels and significantly decreased in human AH specimens. FoxO1 was identified as a transcription factor for MIR148A transactivation. MiR-148a directly inhibited thioredoxin-interacting protein (TXNIP) expression. Consequently, treatment of hepatocytes with ethanol resulted in TXNIP overexpression, activating NLRP3 inflammasome and caspase-1-mediated pyroptosis. These events were reversed by miR-148a mimic or TXNIP small-interfering RNA transfection. Hepatocyte-specific delivery of miR-148a to mice abrogated alcohol-induced TXNIP overexpression and inflammasome activation, attenuating liver injury.Alcohol decreases miR-148a expression in hepatocytes through FoxO1, facilitating TXNIP overexpression and NLRP3 inflammasome activation, which induces hepatocyte pyroptosis. Our findings provide information on novel targets for reducing incidence and progression of ALD.
|Inhibition of MicroRNA-23b Attenuates Immunosuppression in Late Sepsis through NIK, TRAF1 and XIAP.|
Zhang, H; Li, H; Shaikh, A; Caudle, Y; Yao, B; Yin, D
J Infect Dis 0 2018
microRNA-23b (miR-23b) is a multiple functional miRNA. We hypothesize that miR-23b plays a role in the pathogenesis of sepsis. Our study investigated the effect of miR-23b on sepsis induced immunosuppression.Mice were treated with miR-23b inhibitors by tail vein injection 2 days after cecal ligation puncture (CLP)-induced sepsis. Apoptosis in spleens and apoptotic signals were detected, meanwhile survival was monitored. T cell immunoreactivities were examined in late sepsis. NF-κB-inducing kinase (NIK), TNF-receptor associated factor-1 (TRAF1) and X-linked inhibitor of apoptosis protein (XIAP), as the putative targets of miR-23b, were identified by dual luciferase reporter assay.miR-23b expression is upregulated and sustained during sepsis. The activation of TLR4/9/p38/STAT3 signal pathway contributes to the production of miR-23b in CLP-induced sepsis. miR-23b inhibitor decreased TUNEL positive cells in spleen and improved survival. MiR-23b inhibitor restored the immunoreactivity by alleviating the development of T cell exhaustion and producing smaller amounts of immunosuppressive IL-10 and IL-4 in late sepsis. We demonstrated that miR-23b mediated immunosuppression in late sepsis by inhibiting non-canonical NF-κB signal and promoting pro-apoptotic signal pathway via targeting NIK, TRAF1 and XIAP.Inhibition of miR-23b reduces late sepsis-induced immunosuppression and improves survival. miR-23b might be a target for immunosuppression.
|Precise nanoinjection delivery of plasmid DNA into a single fibroblast for direct conversion of astrocyte.|
Park, HS; Kwon, H; Yu, J; Bae, Y; Park, JY; Choi, KA; Choi, Y; Hong, S
Artif Cells Nanomed Biotechnol 0 1-9 2018
Direct conversion is a powerful approach to safely generate mature neural lineages with potential for treatment of neurological disorders. Astrocytes play a crucial role in neuronal homeostasis and their dysfunctions contribute to several neurodegenerative diseases. Using a single-cell approach for precision, we describe here a robust method using optimized DNA amounts for the direct conversion of mouse fibroblasts to astrocytes. Controlled amount of the reprogramming factors Oct4, Sox2, Klf4 and cMyc was directly delivered into a single fibroblast cell. Consequently, 2500 DNA molecules, no more or less, were found to be the optimal amount that dramatically increased the expression levels of the astrocyte-specific markers GFAP and S100b and the demethylation gene TET1, the expression of which was sustained to maintain astrocyte functionality. The converted astrocytes showed glutamate uptake ability and electrophysiological activity. Furthermore, we demonstrated a potential mechanism whereby fibroblast was directly converted into astrocyte at a single-cell level; this was achieved by activating BMP2 pathway through direct binding of Sox2 protein to BMP2 gene. This study suggests that nanotechnology for directly injecting plasmid DNAs into cell nuclei may help understand such a conversion at single-cell level.
|HPSE enhancer RNA promotes cancer progression through driving chromatin looping and regulating hnRNPU/p300/EGR1/HPSE axis.|
Jiao, W; Chen, Y; Song, H; Li, D; Mei, H; Yang, F; Fang, E; Wang, X; Huang, K; Zheng, L; Tong, Q
Oncogene 37 2728-2745 2018
Recent studies reveal the emerging functions of enhancer RNAs (eRNAs) in gene expression. However, the roles of eRNAs in regulating the expression of heparanase (HPSE), an established endo-β-D-glucuronidase essential for cancer invasion and metastasis, still remain elusive. Herein, through comprehensive analysis of publically available FANTOM5 expression atlas and chromatin interaction dataset, we identified a super enhancer and its derived eRNA facilitating the HPSE expression (HPSE eRNA) in cancers. Gain-of-function and loss-of-function experiments indicated that HPSE eRNA facilitated the in vitro and in vivo tumorigenesis and aggressiveness of cancer cells. Mechanistically, as a p300-regulated nuclear noncoding RNA, HPSE eRNA bond to heterogeneous nuclear ribonucleoprotein U (hnRNPU) to facilitate its interaction with p300 and their enrichment on super enhancer, resulting in chromatin looping between super enhancer and HPSE promoter, p300-mediated transactivation of transcription factor early growth response 1 (EGR1), and subsequent elevation of HPSE expression. In addition, rescue studies in HPSE overexpressing or silencing cancer cells indicated that HPSE eRNA exerted oncogenic properties via driving HPSE expression. In clinical cancer tissues, HPSE eRNA was highly expressed and positively correlated with HPSE levels, and served as an independent prognostic factor for poor outcome of cancer patients. Therefore, these findings indicate that as a novel noncoding RNA, HPSE eRNA promotes cancer progression through driving chromatin looping and regulating hnRNPU/p300/EGR1/HPSE axis.
|Long noncoding RNA CCAT2 is activated by E2F1 and exerts oncogenic properties by interacting with PTTG1 in pituitary adenomas.|
Fu, D; Zhang, Y; Cui, H
Am J Cancer Res 8 245-255 2018
Pituitary adenomas, arising from the pituitary gland cells, are one of the most frequent tumors found in the sella region. However, the molecular mechanisms involved in the carcinogenesis and progression of pituitary adenomas is still not understood in detail. Long noncoding RNA (lncRNA) colon cancer-associated transcript 2 (CCAT2), a newly identified lncRNA, has been reported to be abnormally expressed in some cancers. In the present study, we found that CCAT2 was significantly upregulated in pituitary adenomas tissues. Elevated CCAT2 expression was correlated with poor prognosis in patients with pituitary adenomas. Moreover, CCAT2 expression was activated by E2F1. Loss-of-function and gain-of-function assays showed that CCAT2 positively regulated pituitary adenoma cell proliferation, migration, and invasion. Further investigation demonstrated that CCAT2 interacted with PTTG1, and promoted its stability. Furthermore, CCAT2 affected the expression of downstream genes regulated by PTTG1, including SOX2, DLK1, MMP2, and MMP13. Cumulatively, CCAT2 functions as an oncogene in pituitary adenomas and its overexpression contributes to pituitary adenoma carcinogenesis and progression.
|DJ-1 is involved in epigenetic control of sphingosine-1-phosphate receptor expression in vascular neointima formation.|
Lee, KP; Baek, S; Jung, SH; Cui, L; Lee, D; Lee, DY; Choi, WS; Chung, HW; Lee, BH; Kim, B; Won, KJ
Pflugers Arch 0 2018
DJ-1 and sphingosine-1-phosphate (S1P) receptors (S1PRs) are implicated in the control of physiology and pathophysiology of cardiovascular systems such as blood pressure, atherosclerosis, and restenosis. Here, we investigated whether DJ-1 with antioxidant function participates in the regulation of S1PR1 and S1PR2 expression in vascular smooth muscle cells (VSMCs) and whether this response is related to vascular neointima formation. In vitro studies used cellular migration assay, western blot, reverse transcriptase and real-time PCR analysis, and immunocytochemistry. In vivo studies were performed using the carotid artery ligation model together with immunohistochemistry in DJ-1 knockout (DJKO) and corresponding wild-type (DJWT) mice. S1P stimulated migration of VSMCs from DJKO and DJWT mice. VSMC migration was suppressed by S1PR1 inhibitor but was elevated by S1PR2 inhibitor. Compared with DJWT mice, S1PR1 expression was higher in VSMCs and neointimal plaque from DJKO mice, but S1PR2 expression was lower. Overexpression of DJ-1 in DJKO VSMCs reduced S1PR1 expression and elevated S1PR2 expression. Compared with DJWT mice, histone deacetylase-1 recruitment and histone H3 acetylation at the S1PR1 promoter region were lower and higher, respectively, but this pattern was reversed at the S1PR2 promoter region in DJKO VSMCs. S1PR expressions and epigenetic changes at S1PR promoter regions in DJWT VSMCs treated with H2O2 showed similar patterns to those in DJKO VSMCs. Our findings suggest that DJ-1 may be involved in the regulation of S1PR1 and S1PR2 expression via H2O2-mediated histone modification in VSMCs. Consequently, this modification may affect S1P-induced VSMC migration and be related to vascular neointima formation.
|miR-25-3p, Positively Regulated by Transcription Factor AP-2α, Regulates the Metabolism of C2C12 Cells by Targeting Akt1.|
Zhang, F; Chen, K; Tao, H; Kang, T; Xiong, Q; Zeng, Q; Liu, Y; Jiang, S; Chen, M
Int J Mol Sci 19 2018
miR-25, a member of the miR-106b-25 cluster, has been reported as playing an important role in many biological processes by numerous studies, while the role of miR-25 in metabolism and its transcriptional regulation mechanism remain unclear. In this study, gain-of-function and loss-of-function assays demonstrated that miR-25-3p positively regulated the metabolism of C2C12 cells by attenuating phosphoinositide 3-kinase (PI3K) gene expression and triglyceride (TG) content, and enhancing the content of adenosine triphosphate (ATP) and reactive oxygen species (ROS). Furthermore, the results from bioinformatics analysis, dual luciferase assay, site-directed mutagenesis, qRT-PCR, and Western blotting demonstrated that miR-25-3p directly targeted the AKT serine/threonine kinase 1 (Akt1) 3' untranslated region (3'UTR). The core promoter of miR-25-3p was identified, and the transcription factor activator protein-2α (AP-2α) significantly increased the expression of mature miR-25-3p by binding to its core promoter in vivo, as indicated by the chromatin immunoprecipitation (ChIP) assay, and AP-2α binding also downregulated the expression of Akt1. Taken together, our findings suggest that miR-25-3p, positively regulated by the transcription factor AP-2α, enhances C2C12 cell metabolism by targeting the Akt1 gene.
|GATA4 as a novel regulator involved in the development of the neural crest and craniofacial skeleton via Barx1.|
Guo, S; Zhang, Y; Zhou, T; Wang, D; Weng, Y; Chen, Q; Ma, J; Li, YP; Wang, L
Cell Death Differ 0 2018
The role of GATA-binding protein 4 (GATA4) in neural crest cells (NCCs) is poorly defined. Here we showed that mouse NCCs lacking GATA4 exhibited developmental defects in craniofacial bone, teeth, and heart. The defects likely occurred due to decreased cell proliferation at the developmental stage. The in vitro results were consistent with the mouse model. The isobaric tags for relative and absolute quantitation assay revealed that BARX1 is one of the differentially expressed proteins after GATA4 knockdown in NCCs. On the basis of the results of dual-luciferase, electro-mobility shift, and chromatin immunoprecipitation assays, Barx1 expression is directly regulated by GATA4 in NCCs. In zebrafish, gata4 knockdown affects the development of NCCs derivatives. However, the phenotype in zebrafish could be partly rescued by co-injection of gata4 morpholino oligomers and barx1 mRNA. This study identified new downstream targets of GATA4 in NCCs and uncovered additional evidence of the complex regulatory functions of GATA4 in NCC development.
|An Introduction to Antibodies and Their Applications|
|Shaping Epigenetics Discovery - Epigenetics Product Selection Brochure|
|Reprogramming Cell Fate and Function Novel Strategies for iPSC Generation, Characterization, and Differentiation|
|How should I resuspend my pellet prior to PCR?||You should resuspend your pellet in water and not TE as the EDTA found in the TE may interfere with PCR.|
|How many PCR reactions can be done with this kit?||There are enough primers and PCR buffer for 4 reactions per IP assuming a 20 microliter volume and assuming the primers are at the recommended concentration as stated in the manual.|
|Is there ever a time when I do not need to cross-link Histones?||In native ChIP, Histone H3 and Histone H4 do not need to be crosslinked as they are very tightly associated. Histone H2A and Histone H2B are not as tightly associated, but will still work in native ChIP.|
|From where are the primer sequences derived for the kit?||The primer sequences are based on the Human GAPDH promoter. The GenBank number is NT_009759.15, using nts:6497145-6498136.|
|What were your conditions for PCR?||Please see the manual for The EZ ChIP Kit (Catalog #17-371) for more information.|
|If I wanted to quantitate my immunoprecipitated DNA, how would I do so?||DNA purified from ChIP experiments can be quantitated by PCR, providing the amplifying oligos meet specific criteria. Oligos should be 24 mers, with a GC content of 50% (+/- 4) and a Tm of 60.0C (+/- 2.0). You must be certain that the PCR reactions are within the linear range of amplification. Generally it takes time to achieve this. Too much input DNA will affect your results, so set up several tubes for each experiment to optimize the input DNA. Generally, this is about 1/25th to 1/100th for yeast, approximately 1/10 for mammalian cells, but depends on the amount of antibody and input chromatin.
Also, do not use more than 20 cycles, making sure that dNTP's always remain in excess. Also, include each reaction a control primer (to compare your experimental band against-make sure the sizes are sufficiently different to allow proper separation-75 base pairs is usually OK) set to a region of the genome that should not change throughout your experimental conditions. Also PCR from purified input DNA (no ChIP) and include no antibody control PCR's as well. PCR products should be no more than 500 base pairs and should span the area of interest (where you think you will see changes in acetylation or methylation of histones). All PCR products should be run on 7-8% acrylamide gels and stained with SYBR Green 1 (Molecular Probes) at a dilution of 1:10,000 (in 1X Tris-borate-EDTA buffer, pH 7.5) for 30 minutes-no destaining is required.
Quantitation is carried out subsequent to scanning of the gel on a Molecular Dynamics Storm 840 or 860 in Blue fluorescence mode with PMT voltage at 900 with ImageQuant software. This has distinct advantages over ethidium bromide staining. SYBR Green is much more sensitive, and illumination of ethidium stained gels can vary across the gel based on the quality of UV bulbs in your in your light box. For further info, see Strahl-Bolsinger et al. (1997) Genes Dev. 11: 83-93. A radioactive quantitation m
|I am not getting amplification with input DNA. What did I do wrong?||Your input DNA sample should be taken just prior to adding the antibody. It is considered the starting material. If you are not seeing amplification with your input DNA, either you have not successfully reversed the cross links or the PCR is not working for reasons other than the kit.|
|How would you recommend eluting Antibody-protein-DNA complexes from agarose (or sepharose) in order to perform a Re-ChIP experiment?||The complex is removed with the elution buffer that you find in the ChIP assay kit. For a re-CHIP, it might make sense to add protease inhibitors to the IP wash buffers and the elution buffer and the second set of dilution buffers. Make sure everything stays cold so that the proteins aren't degraded during the collection of the first complex or during the second IP.|
|Do you have any tips for sonication?||Keep cells on ice throughout the procedure - even during sonication. Be sure that you don't sonicate for to long (more than 30 seconds could cause sample overheating and denaturation).|
|Why is more DNA is precipitated in my no-antibody control than for my test sample?||To eliminate banding in your negative controls you can do several things:
A) Pre-clear the 2ml diluted cell pellet suspension with 80 microliters of Salmon Sperm DNA/Protein A Agarose-50% Slurry for 30 minutes at 4ºC with agitation. You could try to preclear the lysate longer or with more clearings.
B) Titrate your input DNA, to see when the bands in the NFA disappear.
C) Use an alternative lysis procedure: Resuspend cell pellet in 200 microliters of 5mM Pipes pH 8.0, 85mM KCl, 0.5% NP40 containing protease inhibitors. Place on ice for 10 minutes. Pellet by centrifugation (5 minutes at 5000 rpm). Resuspend pellet in 200 microliters of 1% SDS, 10mM EDTA, 50mM Tris-HCl, pH 8.1 containing protease inhibitors. Incubate on ice for 10 minutes.
D) Block the Salmon Sperm DNA Agarose prior to use in 1-5% BSA and Chip dilution buffer (mix at room temperature for 30 minutes). After incubation, spin the agarose and remove the 1% BSA/ChIP assay buffer supernatant. Wash once in ChIP assay buffer and continue.